Organ transplantation is routinely used to replace entire organs in patients with a variety of health issues, yet there are currently more than 100,000 patients waiting to receive transplants due to lack of donors, and this number is rising dramatically. Exogenic organ production has the potential to alleviate the lack of available organs and help patients who are in desperate need. In this proposal, we aim to develop a facile platform upon which human organs can be grown in a pig, and demonstrate the feasibility of this by generating pig-pig chimeras in which the pancreas and its associated vasculature are entirely derived from donor pig cells. We will be using blastocyst complementation, which has been demonstrated to work in both mouse and pig, and here, we will address some major issues to make this technique clinically applicable. First, we will assess various pluripotent cells that coud be used for donor cells, to overcome the availability, cost and feasibility of using pig or human ES cells and allow us to query a number of developmental genes for exogenic organ production in the pig. Second, we will employ Recombinetics' innovative and proprietary livestock gene editing techniques to quickly and easily ablate the function of multiple genes, allowing the production of chimeric animals with multiple cell lineages being donor-derived. Specifically, two genes that are necessary for the formation of the pancreas and pancreatic vasculature will be knocked out, allowing for the formation of an exogenic pancreas in pigs harboring vessels that are all solely donor-derived. This platform will accelerate the field of exogenic organ production and meets several needs identified in the NIH Symposium for Improving Animal Models for Regenerative Medicine. In follow-on Phase II studies, we will evaluate organogenesis in multi-lineage ablated hosts after complementation with human pluripotent cells.